Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs.

Solubilisation of drugs within liposomal bilayers:alternatives to cholesterol as a membrane stabilising agent

Objectives The aim of this work was to investigate the effect of cholesterol on the bilayer loading of drugs and their subsequent release and to investigate fatty alcohols as an alternative bilayer stabiliser to cholesterol. Methods The loading and release rates of four low solubility drugs (diazepam, ibuprofen, midazolam and propofol) incorporated within the bilayer of multilamellar liposomes which contained a range of cholesterol (0–33 mol/mol%) or a fatty alcohol (tetradecanol, hexadecanol and octadecanol) were investigated. The molecular packing of these various systems was also investigated in Langmuir monolayer studies. Key findings Loading and release of drugs within the liposome bilayer was shown to be influenced by their cholesterol content: increasing cholesterol content was shown to reduce drug incorporation and inclusion of cholesterol in the bilayer changed the release profile of propofol from zero-order, for phosphatidyl choline only liposomes, to a first-order model when 11 to 33 total molar % of cholesterol was present in the formulation. At higher bilayer concentrations substitution of cholesterol with tetradecanol was shown to have less of a detrimental impact on bilayer drug loading. However, the presence of cholesterol within the liposome bilayer was shown to reduce drug release compared with fatty alcohols. Monolayer studies undertaken showed that effective mean area per molecule for a 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) : cholesterol mixture deviated by 9% from the predicted area compared with 5% with a similar DSPC : tetradecanol mixture. This evidence, combined with cholesterol being a much more bulky structure, indicated that the condensing influence of tetradecanol was less compared with cholesterol, thus supporting the reduced impact of tetradecanol on drug loading and drug retention. Conclusions Liposomes can be effectively formulated using fatty alcohols as an alternative bilayer stabiliser to cholesterol. The general similarities in the characteristics of liposomes containing fatty alcohols or cholesterol suggest a common behavioural influence for both compounds within the bilayer.

Interdigitating organic bilayers direct the short interlayer spacing in hybrid organic–inorganic layered vanadium oxide nanostructures

Layered metal oxides provide a single-step route to sheathed superlattices of atomic layers of a variety of inorganic materials, where the interlayer spacing and overall layered structure forms the most critical feature in the nanomaterials’ growth and application in electronics, health, and energy storage. We use a combination of computer simulations and experiments to describe the atomic-scale structure, dynamics and energetics of alkanethiol-intercalated layered vanadium oxide-based nanostructures. Molecular dynamics (MD) simulations identify the unusual substrate-constrained packing of the alkanethiol surfactant chains along each V2O5 (010) face that combines with extensive interdigitation between chains on opposing faces to maximize three-dimensional packing in the interlayer regions. The findings are supported by high resolution electron microscopy analyses of synthesized alkanethiol-intercalated vanadium oxide nanostructures, and the preference for this new interdigitated model is clarified using a large set of MD simulations. This dependency stresses the importance of organic–inorganic interactions in layered material systems, the control of which is central to technological applications of flexible hybrid nanomaterials.

Channel-forming activity of syringopeptin 25 A in mercury-supported phospholipid monolayers and negatively charged bilayers

Interactions of the cationic lipodepsipeptide syringopeptin 25 A (SP25A) with mercury-supported dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS) and dioeleoylphosphatidic acid (DOPA) self-assembled monolayers (SAMs) were investigated by AC voltammetry in 0.1 M KCl at pH 3, 5.4 and 6.8. SP25A targets and penetrates the DOPS SAM much more effectively than the other SAMs not only at pH 6.8, where the DOPS SAM is negatively charged, but also at pH 3, where it is positively charged just as SP25A. Similar investigations at tethered bilayer lipid membranes (tBLMs) consisting of a thiolipid called DPTL anchored to mercury, with a DOPS, DOPA or DOPC distal monolayer on top of it, showed that, at physiological transmembrane potentials, SP25A forms ion channels spanning the tBLM only if DOPS is the distal monolayer. The distinguishing chemical feature of the DOPS SAM is the ionic interaction between the protonated amino group of a DOPS molecule and the carboxylate group of an adjacent phospholipid molecule. Under the reasonable assumption that SP25A preferentially interacts with this ion pair, the selective lipodepsipeptide antimicrobial activity against Gram-positive bacteria may be tentatively explained by its affinity for similar protonated amino-carboxylate pairs, which are expected to be present in the peptide moieties of peptidoglycan strands.

Superlattice structures in twisted bilayers of folded graphene

The electronic properties of bilayer graphene strongly depend on relative orientation of the two atomic lattices. Whereas Bernal-stacked graphene is most commonly studied, a rotational mismatch between layers opens up a whole new field of rich physics, especially at small interlayer twist. Here we report on magnetotransport measurements on twisted graphene bilayers, prepared by folding of single layers. These reveal a strong dependence on the twist angle, which can be estimated by means of sample geometry. At small rotation, superlattices with a wavelength in the order of 10 nm arise and are observed by friction atomic force microscopy. Magnetotransport measurements in this small-angle regime show the formation of satellite Landau fans. These are attributed to additional Dirac singularities in the band structure and discussed with respect to the wide range of interlayer coupling models.

Preferential location of prilocaine and etidocaine in phospholipid bilayers: A molecular dynamics study

In this work, we report a 20-ns constant pressure molecular dynamics simulation of the uncharged form of two amino-amide local anesthetics (LA). etidocaine and prilocaine, present at 1:3 LA:lipid, molar ratio inside the membrane, in the hydrated liquid crystal bilayer phase of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Both LAs induced lateral expansion and a concomitant contraction in the bilayer thickness. A decrease in the acyl chain segment order parameter, -S(CD), compared to neat bilayers, was also observed. Besides, both LA molecules got preferentially located in the hydrophobic acyl chains region, with a maximum probability at similar to 12 and similar to 10 angstrom from the center of the bilayer for prilocaine and etidocaine, respectively. (C) 2009 Elsevier B.V. All rights reserved.

Natural articular joints : model of lamellar-rollerbearing lubrication and the nature of the cartilage surface

The influence of pH on interfacial energy and wettability distributed over the phospholipid bilayer surface were studied, and the importance of cartilage hydrophobicity (wettability) on the coefficient of friction (f) was established. It is argued that the wettability of cartilage signifi antly depends on the number of phospholipid bilayers acting as solid lubricant; the hypothesis was proven by conducting friction tests with normal and lipid- depleted cartilage samples. A lamellar-roller-bearing lubrication model was devised involving two mechanisms: (i) lamellar frictionless movement of bilayers, and (ii) roller-bearing lubrication mode through structured synovial fluid, which operates when lamellar spheres, liposomes and macromolecules act like a roller-bearing situated between two cartilage surfaces in effective biological lubrication.

Binding of small mono- and oligomeric integrin ligands to membrane-embedded integrins monitored by surface plasmon-enhanced fluorescence spectroscopy

We recently developed a binding assay format by incorporating native transmembrane receptors into artificial phospholipid bilayers on biosensor devices for surface plasmon resonance spectroscopy. By extending the method to surface plasmon-enhanced fluorescence spectroscopy (SPFS), sensitive recording of the association of even very small ligands is enabled. Herewith, we monitored binding of synthetic mono- and oligomeric RGD-based peptides and peptidomimetics to integrins alphavbeta3 and alphavbeta5, after having confirmed correct orientation and functionality of membrane-embedded integrins. We evaluated integrin binding of RGD multimers linked together via aminohexanoic acid (Ahx) spacers and showed that the dimer revealed higher binding activity than the tetramer, followed by the RGD monomers. The peptidomimetic was also found to be highly active with a slightly higher selectivity toward alphavbeta3. The different compounds were also evaluated in in vitro cell adhesion tests for their capacity to interfere with alphavbeta3-mediated cell attachment to vitronectin. We hereby demonstrated that the different RGD monomers were similarly effective; the RGD dimer and tetramer showed comparable IC50 values, which were, however, significantly higher than those of the monomers. Best cell detachment from vitronectin was achieved by the peptidomimetic. The novel SPFS-binding assay platform proves to be a suitable, reliable, and sensitive method to monitor the binding capacity of small ligands to native transmembrane receptors, here demonstrated for integrins.

Computational model of membrane fission catalyzed by ESCRT-III

ESCRT-III proteins catalyze membrane fission during multi vesicular body biogenesis, budding of some enveloped viruses and cell division. We suggest and analyze a novel mechanism of membrane fission by the mammalian ESCRT-III subunits CHMP2 and CHMP3. We propose that the CHMP2-CHMP3 complexes self-assemble into hemi-spherical dome-like structures within the necks of the initial membrane buds generated by CHMP4 filaments. The dome formation is accompanied by the membrane attachment to the dome surface, which drives narrowing of the membrane neck and accumulation of the elastic stresses leading, ultimately, to the neck fission. Based on the bending elastic model of lipid bilayers, we determine the degree of the membrane attachment to the dome enabling the neck fission and compute the required values of the protein-membrane binding energy. We estimate the feasible values of this energy and predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission. We support the computational model by electron tomography imaging of CHMP2-CHMP3 assemblies in vitro. We predict a high efficiency for the CHMP2-CHMP3 complexes in mediating membrane fission.

Molecular Origin of the Intrinsic Bending Force for Helical Morphology Observed in Chiral Amphiphilic Assemblies: Concentration and Size Dependence

The formation of the helical morphology in monolayers and bilayers of chiral amphiphilic assemblies is believed to be driven at least partly by the interactions at the chiral centers of the amphiphiles. However, a detailed microscopic understanding of these interactions and their relation with the helix formation is still not clear. In this article a study of the molecular origin of the chirality-driven helix formation is presented by calculating, for the first time, the effective pair potential between a pair of chiral molecules. This effective potential depends on the relative sizes of the groups attached to the two chiral centers, on the orientation of the amphiphile molecules, and also on the distance between them. We find that for the mirror-image isomers (in the racemic modification) the minimum energy conformation is a nearly parallel alignment of the molecules. On the other hand, the same for a pair of molecules of one kind of enantiomer favors a tilt angle between them, thus leading to the formation of a helical morphology of the aggregate. The tilt angle is determined by the size of the groups attached to the chiral centers of the pair of molecules considered and in many cases predicted it to be close to 45 degrees. The present study, therefore, provides a molecular origin of the intrinsic bending force, suggested by Helfrich (J. Chem. Phys. 1986, 85, 1085-1087), to be responsible for the formation of helical structure. This effective potential may explain many of the existing experimental results, such as the size and the concentration dependence of the formation of helical morphology. It is further found that the elastic forces can significantly modify the pitch predicted by the chiral interactions alone and that the modified real pitch is close to the experimentally observed value. The present study is expected to provide a starting point for future microscopic studies.

Chiral molecular self-assembly of phospholipid tubules: A circular dichroism study

We report on spectroscopic studies of the chiral structure in phospholipid tubules formed in mixtures of alcohol and water. Synthetic phospholipids containing diacetylenic moieties in the acyl chains self-assemble into hollow, cylindrical tubules in appropriate conditions. Circular dichroism provides a direct measure of chirality of the molecular structure. We find that the CD spectra of tubules formed in mixtures of alcohol and water depends strongly on the alcohol used and the lipid concentration. The relative spectral intensity of different circular dichroism bands correlates with the number of bilayers observed using microscopy. The results provide experimental evidence that tubule formation is based on chiral packing of the lipid molecules and that interbilayer interactions are important to the tubule structure

X-Ray Studies On The Bilayer Structure Of Trypsin-Treated Rat-Brain Myelin

Trypsin-treated rat brain myelin was subjected to biochemical and X-ray studies. Untreated myelin gave rise to a pattern of three rings with a fundamental repeat period of 155 Angstrom consisting of two bilayers per repeat period, whereas myelin treated with trypsin showed a fundamental repeat period of 75 Angstrom with one bilayer per repeat period. The integrated raw intensity of the h=4 reflection with respect to the h=2 reflection is 0.38 for untreated myelin. The corresponding value reduced to 0.23, 0.18, 0.17 for myelin treated with 5, 10, 40 units of trypsin per mg of myelin, respectively, for 30 min at 30 degrees C. The decrease in relative raw intensity of the higher-order reflection relative to the lower-order reflection is suggestive of a disordering of the phosphate groups upon trypsin treatment or an increased mosaicity of the membrane or a combination of both these effects, However, trypsin treatment does not lead to a complete breakdown of the membrane, The integrated intensity of the h=1 reflection, though weak, is above the measurable threshold for untreated myelin, whereas the corresponding intensity is below the measurable threshold for trypsin-treated myelin, indicating a possible asymmetric to symmetric transition of the myelin bilayer structure about its centre after trypsin treatment.

Synthesis and vesicle formation from hybrid bolaphile/amphiphile ion-pairs. Evidence of membrane property modulation by molecular design

Four new hybrid (bolaphile/amphiphile) ion-pairs were synthesized. Electron microscopy indicated that each of these forms bilayer membranes upon dispersion in aqueous media. Membrane properties have also been examined by differential scanning calorimetry, microcalorimetry, temperature-dependent fluorescence anisotropy measurements, and UV-vis spectroscopy. The T-m values for the vesicular 1, 2, 3, 4, and 5 were 38, 12, 85, 31.3, and 41.6 degrees C, respectively. Interestingly the T-m values for 1 and 3 were found to depend on their concentration. The entrapment of small solute and the release capability have also been examined to demonstrate that these bilayers form enclosed vesicles. X-ray diffraction of the cast films has been performed to understand the nature and the thickness of these membrane organizations. The membrane widths ranged from 33 to 47 Angstrom. Finally, the above observations have been analyzed in light of the results obtained from molecular modeling studies. Thus we have demonstrated that membrane properties can be modulated by simple structural changes at the amphiphile level. It was shown that by judicious incorporation of central, isomeric, disubstituted aromatic units as structural anchors into different bolaphiles, one can modulate the properties of the resulting vesicles.

Synthesis of macrocyclic diacyl/dialkyl glycerols containing disulfide tether and studies of their effects upon incorporation in DPPC membranes. Implications in the design of phospholipase A(2) modulators

A general method for the preparation of novel disulfide-tethered macrocyclic diacylglycerols (DAGs) has been described. Overall synthesis involved stepwise protection, acylation, and deprotection to yield the bis(omega-bromoacyl) glycerols. In the crucial macrocyclization step, a unique reagent, benzyltriethylammonium tetrathiomolybdate (BTAT), has been used to convert individual bis(omega-bromoacyl) glycerols to their respective macrocyclic disulfides. DAG 6, which had ether linkages between hydrocarbon chains and the glycerol backbone, was also synthesized from an appropriate precursor using a similar protocol. One of the DAGs (DAG 5) had a carbon-carbon tether instead of a disulfide one and was synthesized using modified Glaser coupling. Preparation of alpha-disulfide-tethered DAG (DAG 4) required an alternative method, as treatment of the bisbromo precursor with BTAT gave a mixture of several compounds from which separation of the target molecule was cumbersome. To avoid this problem, the bisbromide was converted to its corresponding dithiocyanate, which on further treatment with BTAT yielded the desired DAG (DAG 4) in good yield. Upon treatment with the reducing agent dithiothreitol (DTT), the DAGs that contain a disulfide tether could be quantitatively converted to their "open-chain" thiol analogues. These macrocyclic DAGs and their reduced "open-chain" analogues have been incorporated in DPPC vesicles to study their effect on model membranes. Upon incorporation of DAG 1 in DPPC vesicles, formation of new isotropic phases was observed by P-31 NMR, These isotropic phases disappeared completely on opening the macrocyclic ring by a reducing agent. The thermotropic properties of DPPC bilayers having DAGs (1-6) incorporated at various concentrations were studied by differential scanning calorimetry. Incorporation of DAGs in general reduced the cooperativity unit (CU) of the vesicles. Similar experiments with reduced "open-chain" DAGs incorporated in a DPPC bilayer indicated a recovery of CU with respect to their macrocyclic "disulfide" counterparts. The effect of inclusion of these DAGs on the activity of phospholipase A(2) (PLA(2)) was studied in vitro. Incorporation of DAC 1 in DPPC membranes potentiated both bee venom and cobra venom PLA(2) activities.

Vortex dynamics at rf frequencies in Bi2Sr2CaCu2O8 single crystals

We have studied the low magnetic field high temperature region of the H-T phase diagram of Bi2Sr2CaCu2O8 single crystals using the technique of non-resonant rf response at a frequency of 20 MHz. With H(rf)parallel to a, H parallel to c, the isothermal magnetic field scans below T-c show that the frequency f(H) of the tank circuit decreases continuously with increase in H before saturating at H similar to H-D(T). Such a decrease in f(H) reflects increasing rf penetration into the weakly screened region between CuO bilayers. The saturation of f(H) at its lowest value for H similar to H-D(T) indicates complete rf penetration land hence the disappearance of field dependence) due to the vanishing of the screening rf currents I-rf(c) in those regions or equivalently when the phase coherence between adjacent superconducting layers vanishes. Therefore H,(T) represents the decoupling of the adjacent superconducting bilayers, and hence also a 3D to 2D decoupling transition of the vortex structure. Simultaneous monitoring of the field dependent rf power dissipation P(H) shows a maximum in dP/dH at H-D(T). The observed H-D(T) line in many crystals is in excellent agreement with the (l/t-1) behavior proposed for decoupling.